The global increase in livestock and poultry production observed in the last decades has led to an increase in animal waste generated. The animal waste contains high levels of nitrogen and may carry antibiotics that can disturb important microbial activities such as denitrification in terrestrial and aquatic ecosystems. Disturbances of microbial denitrification can have detrimental consequences to environmental health. In the terrestrial environment, denitrification is an important source and sink of N2O, a potent greenhouse gas in the atmosphere. In aquatic ecosystems, denitrification is a dominant NO3- removal pathway, contributing to prevent eutrophication. The overall goal of this dissertation is to evaluate the impacts of animal waste and potential antibiotic exposure on microbial communities responsible for denitrification in terrestrial and aquatic ecosystems. To achieve this goal, a combined approach of measuring activity rates and performing a molecular characterization of the microbial communities was used. In Chapter 2, the microbial community changes associated with the impacts of acute antibiotic exposure on denitrification were evaluated in soil microcosms. Antibiotic exposure caused a significant increase in N2O production from denitrification. This increase was paralleled with a greater ratio of fungi:bacteria abundance and lower abundances of particular taxa with N2O reduction capacity. In Chapter 3, the impacts of animal manure and antibiotic contamination on N2O fluxes and the abundance of denitrification genes were investigated in soil mesocosms. N2O fluxes in soils treated with manure fertilizer and tetracycline were considerably higher than in control soils. The manure fertilization and antibiotic exposure had diverse effects on different bacterial taxa responsible for N2O production. In Chapter 4, the denitrification activity and microbial community structure in tidal creek sediments impacted by wastewater discharge from a poultry processing plant were evaluated through a field survey and a microcosm experiment. Denitrification rates were inhibited in the location affected by the wastewater discharge. This decrease in denitrification activity was associated with changes in the microbial community structure, such as a lower relative abundance of bacterial taxa carrying denitrification genes and lower abundance of N2O reducing bacteria. In Chapter 5, the abundance and diversity of antibiotic resistance genes were evaluated in a tidal creek impacted by wastewater discharge from a poultry processing plant. The numbers of antibiotic resistance genes were higher in the location closer to the wastewater discharge, suggesting an historic antibiotic exposure associated with the activity of the poultry processing plant. Overall, this work provides new knowledge of the impacts of animal waste and antibiotics on N2O emissions in terrestrial ecosystems and microbial NO3- removal in aquatic ecosystems. This dissertation emphasizes the functional importance of microbial communities to ecosystem health and their responses to anthropogenic disturbance.

Microbial communities are ubiquitous in every ecosystem on earth, but the social interactions within these communities have only recently become a topic of investigation. Biofilms, the most common growth pattern found in nature, offer an exciting opportunity to study these interactions in a complex, spatially structured environment. This series of investigations explored the relationships between chemical warfare, a common competitive strategy, the three-dimensional spatial structure found in biofilms, and the phenotypic variation common in natural communities using Saccharomyces cerevisiae as a model organism. In the first set of experiments, the competitive effects of toxin production and biofilm formation were measured in well-mixed liquid environments, simple agar colonies, and complex biofilm colonies. Spatial structure strongly influenced the final community composition, with significant differences found between the liquid and agar substrates in the majority of cases. Toxin proved to be most effective against competing strains in spatially structured environments, regardless of biofilm formation. In the second set of experiments, the effect of adding a third, toxin-resistant strain was assayed in the same sets of growth conditions. It was expected that in spatially structured conditions, the resistant strain would passively form a physical barrier between the sensitive and toxin-producing strains, thus increasing the fitness of the sensitive strain. The results from these trials indicated that the relationships within these three-strain communities were much more complex than in two-strain communities. The resistant strain acted primarily as a competitor in all cases, and variation in the competitive outcomes of the trials was high. These results suggested when studying communities with multiple lineages, a combination of cell-level fitness metrics and whole-colony spatial analysis is a more appropriate analysis framework. These investigations contributed to an area of study with high ecological and evolutionary relevance but little existing knowledge, and opened new avenues for future research concerning microbial communities.

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The heterotrophic dinoflagellates Pfiesteria piscicida and Pseudopfiesteria shumwayae (here referred to as Pfiesteria) have been reported to secrete potent toxins responsible for inducing lesions in Atlantic menhaden, causing deaths of fishes in natural systems and laboratory assays and impacting human health. Many aspects of Pfiesteria biology and ecology have been questioned, including its complicated life-cycle, its role in fish kills, fish lesion events and human health effects as well as its ability to produce toxins. Although the involvement of Pfiesteria in major estuarine fish kills and lesion events has been disputed, there is a demonstrated ability of these organisms to cause fish pathology and mortality in laboratory bioassays. Little evidence exists however, to support the conclusion that these effects are due to a potent exotoxin. Many other dinoflagellates are morphologically and genetically similarity to Pfiesteria and have been referred to as "Pfiesteria -like", although the pathogenic ability of these other species has not been investigated. In this work we address various aspects of the behavior and biology of Pfiesteria and related dinoflagellates in order to determine their ability to impact the health of fishes and to determine if any adverse effects are caused by a toxin or if of some other pathogenic mechanism is involved. Aquarium-format bioassays commonly used to detect and determine the toxin-producing status of Pfiesteria are subject to impediments that can make determination of the cause of fish mortality difficult. We developed a sensitive, 96 hr larval fish bioassay for assessing Pfiesteria pathogenicity using 2-10 mL volumes and larval cyprinodontid fishes. This assay was a highly effective method to verify and evaluate ichthyocidal activity in these dinoflagellates. Results from the larval assays, together with histopathological analysis, electron microscopy and direct observations of the interactions of Pfiesteria and fish, demonstrated the ability of dinoflagellates of the Pfiesteriaceae (including several Pfiesteria-like species) to cause pathology and mortality by feeding on the epidermal tissues of live fish. For some species, this process of micropredatory feeding resulted in fish mortality in laboratory bioassays comparable to that reported for purportedly "toxic" strains, but without the involvement of a toxin. Thus, the assertion that Pfiesteria produces a potent, fish-killing toxin is disputed. In laboratory assays with larval fish, P. shumwayae was consistently and significantly more ichthyocidal than P. piscicida. This differential pathogenicity was observed to be unrelated to chemoattraction of the dinoflagellates to fish tissues. Instead, as determined with comparative morphometric analyses, differences in ichthyocidal activity between these two species were related to their capacity to grow and reproduce after feeding on fish or algal prey. The results of these studies suggest that the perception of toxicity in these dinoflagellates is erroneous and related to different life history strategies and associated differences in growth rate in response to prey type. There is no direct evidence that these heterotrophic dinoflagellates impact fish health in the natural environment.

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Sterilization methods are typically studied to prove their reliability and predictability. Reliability implies definition of the principle variables affecting sterilization. Predictability suggests the ability to forecast assurance of sterility. Filtration, using membrane filters, is the least well characterized sterilization method with respect to reliability and predictability. Pseudomonas diminuta ATCC 19146, selected as a biological indicator, was optimized for its resistance to removal by filtration. Filtration equipment was designed to allow systematic study of experimental variables. Test methods were developed to allow quantitation of bacterial retention and to define the effects of several parameters (e.g., bacterial numbers, filtration pressure, time and fluid chemistry) on retention. These methods were also used to measure the prediction of retention by a physical test of membrane filters. The impact of experimental variables studied was a function of filter pore size. Specifically, bacterial retention by membrane filters commonly used for sterilization was independent of bacterial numbers, filtration pressure and fluid chemistry and dependent on time. Sterility of filter effluents was consistently achieved by a single layer of a 0.22 (mu)m pore size filter for continuous filtrations up to 16 hours long. The bubble point of membrane filters was a strong predictor of bacterial retention. No bacterial passage was observed above a minimum bubble point value. Microscopic examination of the penetration of bacterial cells as a function of depth within a filter suggested a model of bacterial retention based on a sequential sieving of cells by the three dimensional structure of a filter. The knowledge of filter removal ability was combined with the volume and microbial content of liquids to calculate the probability of sterility assurance. Sterilization by filtration was found to be a reliable and predictable method when applied under properly controlled conditions.

Volcanic activity creates new landforms that can change dramatically as a consequence of biotic succession, and microbes are essential contributors to successional development. Our objective was to expand our knowledge of the spatial and temporal dynamics of microbial communities in nascent soils. To study primary succession we characterized the microbial diversity on a chronosequence of volcanic deposits ranging from 20 to 300 yr located in the Kilauea Volcano, Hawaii by analysis of Bacteria and Archaea 16S rRNA gene sequences amplified from total DNA, Community-Level Phospholipids Fatty Acid, Community-Level Physiological Profiles using ECOplate, and bacterial isolates. A parallel investigation of the extent of secondary succession was made on a nearby geothermally active site. Primary succession. phylogeny of 16S rRNA gene sequences indicated a high diversity of sequences not related to known taxa with 15 classes within the Bacteria domain and a high relative abundance within the Archaea domain of various unclassified non-thermophilic Crenarchaeota. Bacterial richness and diversity increased significantly with age, while no correlation was found among the archaeal community. The 194 isolates, together encompassing only 1.6% of total culture independent diversity, were not among the dominant clones in the libraries. Carbon utilization profiles and plate counts indicated that heterotrophic communities that are established on older sites were more active and occurred in higher numbers. Multivariate analyses showed not only that the bacterial communities of distinct sites and ecosystem regime shared similar phylotypes, but also revealed a gradual succession of the community structure. Secondary succession. elevated soil temperature (up to 87°C), and steam vents provide evidence of an active geothermal system. Bacterial clones and thermophilic Crenarchaeota were limited to the geothermal system, and not detected in the surrounding area. This not only indicates that the temperature shift resulted in a change of the community structure of these volcanic deposits, but also that the underlying strata might be the source for hyperthermophiles. In general, microbes are able to colonize and establish a community among recent volcanic deposits. However, environmental parameters rather than site age influence this successional development. This work yields new insights into survival and succession of microbes in soils.

Dissimilatory iron-reduction in hyperthermophilic (Topt > 80°C) Archaea appears to be a ubiquitous, conserved, and possibly ancient metabolism that remains largely unknown. This investigation sought to understand the physiological and ecological context for iron respiration by the hyperthermophilic Pyrobaculum which are commonly found in terrestrial hydrothermal environments. Iron respiration in Pyrobaculum is differentiated from Bacteria by the lack of polyheme c-type cytochromes common for the mesophiles Geobacter and Shewanella , representing a novel model system for studying iron-reducing microorganisms. The overall change in protein pattern and abundance coupled with specific activities of respiratory enzymes such as the nitrate reductase and ferric reductase support the notion that iron respiration in Pyrobaculum is a regulated process. In Pyrobaculum, the mechanism for electron transfer to insoluble iron oxides seems to vary as P. aerophilum and P. arsenaticum could reduce iron oxide hydroxide when sequestered by semi-permeable dialysis membrane tubing while P. islandicum and P. calidifontis required direct contact with iron oxide hydroxide. Iron-grown membrane fractions of P. aerophilum only were capable of enzymatic oxidation of the reduced soluble quinone analog 2,6-anthrahydroquinone disulfonate (AHDS), while iron-grown membranes from P. islandicum did not show this activity. This suggests that although the ability to reduce iron oxide hydroxides to generate energy is highly conserved within Pyrobaculum , the strategies may not be. Pyrobaculum species are metabolically diverse microorganisms capable of iron, sulfur, thiosulfate, nitrate, and microaerophilic respiration. To understand how anaerobic respiration and the environment may interact, a range of Eh and pH values were investigated to determine their effect on growth rates in Pyrobaculum. Growth rates on sulfur compounds were highest at slightly acid pHs and highly reduced (< -420 mV) conditions while growth rates on nitrate and iron were highest at neutral to slightly basic pHs and more oxidized (> -210 mV) conditions. Almost all known hyperthermophiles in pure culture to date have been isolated with an electron acceptor other than Fe(III). Therefore, it seems likely that a better understanding towards the diversity, physiology, and biogeochemistry of these hydrothermal microbial communities is likely to be gained by directly isolating hyperthermophiles using Fe(III). Two novel microbes were isolated, purified, and characterized during the course of this study from differing hydrothermal sediments. The first organism originated from a deep-sea hydrothermal vent from the northeast Pacific Ocean and is an obligate iron-reducing autotroph named Geogemma pacifica. The second organism investigated was named Pyrobaculum strain SP4 and was isolated from the Caribbean island, St. Lucia. These isolates support the notion that dissimilatory iron reduction is common in these environments and further expands the known locations where Pyrobaculum species have been isolated thus far.